Toxins, chemical compounds produced by living organisms, negatively affect the health of people and animals throughout the world, with the results ranging from mildly disabling disease to immediate death. In fact, one of the most deadly compounds currently known to man is the botulinum toxin; a bacterial toxin produced by the organism Clostridium botulinum. The targets and affects of toxins vary widely and include hemotoxins, which target and destroy red blood cells, necrotoxins, which indiscriminately cause cell death and destruction of all tissue types, and neurotoxins, which target and affect the nervous system. Furthermore, toxin weapons utilized in a terrorist attack could potentially cause mass casualties if properly deployed. Currently, the most effective treatment for toxicity resulting from toxin exposure is the administration of an antitoxin. However, the development of antitoxins can be a time consuming process, and during an attack with a toxin weapon antitoxin stockpiles could become rapidly depleted, thereby leaving effective treatment in short supply. Thus, there exists an immediate need for a treatment that can reduce the toxicity of a toxin, and for methods of identifying new compounds that can combat the threat of an attack with a toxin weapon.
Infectious diseases also affect the health of people and animals around the world, causing serious illness and death. Black Plague devastated the human population in Europe during the middle ages. Pandemic flu killed millions of people in the 20th century and is a threat to reemerge.
Addition of ε-toxin to MDCK cells leads to the formation of detergent-resistant toxin oligomers (Miyata et al., Nagahama et al. (1998), Petit et al.) Formation of the oligomeric complexes is detectable as early as 15 minutes after toxin addition to MDCK cells, at which time 10 to 20% of the monolayer has been killed (Petit et al.) Formation of these oligomeric complexes is observed when ε-toxin is added to sensitive, but not resistant cell lines (Nagahama et al. (1998)). In addition, the active form of ε-toxin, but not the inactive prototoxin, is able to form the detergent-resistant complexes (Nagahama et al. (1998)). Specifically, removal of a carboxyterminal peptide from the ε-prototoxin upon activation is required for both the increased cytotoxicity and the ability to form oligomeric complexes (Miyata et al.) Treating MDCK cells with ε-toxin is rapidly followed by efflux of intracellular K+ and increases in intracellular Cl− and Na+ (Petit et al.) There is no evidence that the ε-toxin enters cells (Nagahama et al. (1998), Petit et al.). Thus, in one pathway, the lethal activity of the toxin can be a direct effect of the toxin forming oligomeric pores in the plasma membrane of target cells, leading to depolarization of the cell's electrochemical gradient, disruption of ion homeostasis, and cell death. However, an alternate pathway leading to cell death also can be involved. Addition of ε-toxin to a murine renal cortical collecting duct cell line leads to a rapid depletion of cellular ATP levels, stimulates AMP-activated protein kinase, and induces nuclear translocation of apoptosis-inducing factor, a potent caspase-independent cell death effector (Chasin et al.) In this study, the ATP-depletion and cell death appeared to be independent of toxin oligomerization and the formation of pores (Chasin et al.) Thus host factors, in addition to the cell-surface receptor, can contribute to ε-toxin-mediated cytotoxicity.
A variety of studies exploring the cytotoxic activities of other pore-forming toxins has suggested that host factors (beyond cell-surface receptors) also contribute to toxin-induced cytotoxicity (Gonzalez et al. Bischof et al., Gurcel et al., Huffman et al., Bellier et al., Zhang et al., Skals et al., Soletti et al.) For example, the mammalian protein kinase A pathway has been shown to be required for Cry 1 Ab-induced cell death (Zhang et al.) Additionally, E. coli α-hemolysin has been shown to lead to leakage of ATP from cells; the extracellular ATP then activates P2X pores that potentiate cell lysis (Skals et al.) Finally, pre-treatment of glioma cells with inhibitors of mitogen-activated/extracellular regulated kinase 1, protein kinase C, or Ca2+/calmodulin-dependent kinase 11 protects cells from Bc2 and equinatoxin-11 (Soletli et al.)
Clostridium difficile infection (CDI), commonly referred to as “C. difficile” or “c-diff”, has become a significant medical problem in hospitals, long-term care facilities, and in the community and is estimated to afflict more than 450,000 people each year in the U.S. Patients typically develop CDI from the use of broad-spectrum antibiotics that disrupt normal gastrointestinal (gut) flora, thus allowing C. difficile bacteria to flourish and produce toxins.
Clostridium difficile is a member of a family of bacteria that are capable of producing toxins in response to environmental stress and can become spores impervious to most means of simple eradication. The toxins produced by these bacteria are the major reason for these organisms being highly pathogenic. At present, between 1.1 and 3 billion dollars are spent in the USA market due to antibiotic induced expression of the C-diff toxins, with direct treatment accounting for at least 10% of the total expenditures.
C-diff produces two toxins of note, TcdA and TcdB (the A and B toxins, respectively). A new strain of c-diff produces 16 to 30 times the amount of toxin A and B as the previously common c-diff isolates, and this new strain accounts for non-hospital based severe infection.
Traditional treatments for toxicity include pharmaceuticals. However, the vast majority of toxins lack an effective drug. Those drugs that exist have several limitations and drawbacks that including limited effectiveness and toxicity. Thus, an urgent need exists for alternative treatments for toxins and infectious diseases, and methods of identifying new drugs to combat these threats.